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Coupled Transcription and Translation Within
Nuclei of Mammalian Cells
Francisco J. Iborra,1 Dean A. Jackson,2 Peter R.
Cook1
Science, Vol. 293, Issue 5532, 1139-1142, August
10, 2001
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Fig. 1. Protein synthesis in permeabilized HeLa
cells and isolated nuclei. (A and B) The effects
of inhibitors on 3Hlysine incorporation. cam,
cx, p 0.3, 1, or 0.3 mg/ml chloramphenicol,
cycloheximide, or puromycin. (C) 3HLeu
incorporation in the presence of
biotin-lysine-tRNA. (D) Sizes of nascent
proteins. Proteins made (after 0, 2, 5, and 20
min) by permeabilized cells or nuclei in 35SMet
were run on a gel (four lanes on left or right,
respectively), stained with Coomassie blue, and
photographed, and an autoradiogram was prepared
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Fig. 2. Biotin peptides light microscopy.
Permeabilized HeLa cells (A to F) and isolated
nuclei (G and H) were allowed to extend nascent
proteins in biotin-lysine-tRNA, sites containing
biotin were indirectly immunolabeled, and single
equatorial sections through nuclei were collected
by "confocal" microscopy. Scale bars, 10 (left)
and 5 (right) µm. (A and B) Zero time control
mitochondria contain high levels of endogenous
(covalently bound) biotin, whereas nuclei contain
background levels. (C and D) Extension for 10
min nuclei and cytoplasm contain more biotin,
and nuclear labeling is concentrated in discrete
sites. (E and F) Extension for 10 min in
cycloheximide (1 mg/ml) most nuclear labeling is
abolished. (G and H) Extension for 10 min,
isolated nuclei most cytoplasm has been lost,
and nuclear foci appear sharper because they
aggregate during isolation. (I) Intensities of
nucleoplasmic labeling in gt100 cells like those
in (C) or (G) were measured, and numbers of cells
or nuclei with intensities (arbitrary units) of 0
to 10, 10 to 20, etc. are plotted. Gray boxes,
permeabilized cells open boxes, isolated nuclei
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Fig. 3. BODIPY peptides light microscopy. (A to
D) HeLa cells were permeabilized, incubated (10
min) BODIPY-Lys-tRNA cycloheximide (1 mg/ml),
fixed, and imaged. Scale bars, 10 (left) and 5
(right) µm. (E) Intensities (arbitrary units) of
nuclear and cytoplasmic regions of gt100 cells at
different times. Autofluorescent background was
10 of the nucleoplasmic signal at 2 min, and
treatment with RNase A (100 µg/ml 30 min 37C)
or proteinase K (500 µg/ml 30 min 30C) reduced
intensities by 5 or 98, respectively
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Fig. 4. Coupled transcription and translation.
(A) Cells were permeabilized and incubated (10
min) with biotin-lysine-tRNA supplements shown,
and sites containing biotinylated polypeptides
were imaged (as in Fig. 2D) then the average
fluorescence intensity over the nucleoplasm in
gt100 cells was measured and expressed relative to
that found without supplements. In the first
experiment (rows 1 to 4) with HeLa (subtetraploid
human carcinoma), Cos-1 (SV40-transformed monkey
cell), MRC5 (diploid human fibroblast), and T-24
(human bladder carcinoma) cells, increasing NTPs
increases nucleoplasmic labeling, but not if
-amanitin (10 µg/ml) is present. In the second
experiment with HeLa (rows 5 to 10), adding 0.5
mM NTPs increases nucleoplasmic labeling, but not
if CTP is omitted or 1 mM 3'dATP (cordycepin
triphosphate) is present. Adding cycloheximide
(cx 1 mg/ml) with (row 9)--or 3 min before (row
10)--biotin-lysine-tRNA also decreases labeling.
, difference significant at 99.9 level
(Student's t test). (B) HeLa cells were
permeabilized and incubated with 3HLys or
BODIPY-Lys-tRNA supplements, and the tagged
peptides were detected by autoradiography or
direct imaging. Average numbers of grains (open
boxes) or fluorescence intensities (gray boxes)
of gt50 cells are expressed relative to values
found in row 1. , difference compared with value
in row 5 significant at 99.99 level (Student's t
test).
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Fig. 5. Biotin peptides electron microscopy. (A)
Permeabilized HeLa cells were incubated (10 min)
in biotin-lysine-tRNA, and sites containing
biotin were indirectly immunolabeled with 10-nm
gold particles most particles are found over
ribosome-rich regions of the cytoplasm, but some
are in clusters over the nucleoplasm. Incubation
with cycloheximide (1 mg/ml) reduced
nucleoplasmic labeling by 92. Scale bar, 200 nm.
(B) Permeabilized cells were incubated (10 min)
in biotin-lysine-tRNA and Br-UTP, and sites
containing biotin-polypeptides and Br-RNA were
indirectly immunolabeled with 5- and 10-nm gold
particles, respectively the two labels lie close
to each other. (C) L7 marked by 10-nm particles
and biotin polypeptides by 5-nm particles. Scale
bar, 50 nm. (D) Observed versus random
colocalization the significance of the fraction
of biotin peptides colocalizing with various
markers was determined by superimposing a grid of
40-nm squares on images like those in (B) and
(C), counting the fraction of squares containing
both types of particles, and comparing the
results with those expected of a Poisson
distribution. , probability that association
arose by chance lt 0.001 (P2 test).